Chondrocytes are specialised cells found in cartilage. Chondrocytes in cartilage produce a large amount of extracellular matrix which is composed of collagen fibers, ground substance, which is rich in proteoglycan, and elastin fibers. The cartilage tissue performs a structural and mechanical function in skeletal joints and any disease or injury is consequently debilitating for patients.
Cartilage degradation is a hallmark of two disease groups: osteoarthritis, a degenerative condition, and rheumatoid arthritis, which is primarily caused by inflammation. The degradation leads to joint pain and mobility impairment to a degree that can be disabling.
Considerable progress has been made in the development of anti-inflammatory agents effective for inhibiting the progress of such diseases. However, where degradation or injury has already taken place, new therapies are needed to assist in the regenerating of joint cartilage.
In the field of regenerative medicine, efforts have been directed at developing cell populations capable of repairing cartilage. Established lines of articular chondrocytes have been described in WO 96/18728 and methods for chondrocyte growth and differentiation have been reported in WO 98/55594. WO 00/27996 reports serum-free medium for chondrocyte like cells, comprising minimum essential medium, growth factors, lipids and amino acids. U.S. Pat. No. 6,150,163 outlines chondrocyte media formulations and culture procedures, in which dedifferentiated human articular chondrocytes are grown in a medium containing TGFβ and either insulin or insulin-like growth factor. It has also been reported that primary chondrocytes cultured in vitro will de-differentiate if not treated with the appropriate factors (Benya et al. Cell 1982 30:215). The cells that are produced are fibroblastic in appearance and may be MSC-like.
Jorgensen et al. (Ann. Rheum. Dis. 60:305, 2001) reviews recent progress in stem cells for repair of cartilage and bone in arthritis. Jakob et al. (J. Cell. Biochem. 26:81, 2001) studied specific growth factors involved in expansion and redifferentiation of adult human articular chondrocytes that enhance chondrogenesis and cartilage formation. M. Brittberg (Clin. Orthop. 367 Suppl:S147, 1999) reviews current chondrocyte transplantation procedures in which pure chondrocytes or other mesenchymal cells are harvested autologously or as allografts from a healthy tissue source, expanded in vitro, and then implanted into the defect at high density.
Despite the initial success of the clinical methods reported to date, it is clear that current sources of chondrocytes are inadequate to treat most of the instances of cartilage degeneration that present themselves at the clinic. In addition, problems concerning the use of immunosuppressive drugs have complicated the success of developing new transplantation protocols.
Regenerative medicine is also benefiting from recent advances relating to the isolation, culture, and use of various types of progenitor cells. Embryonic stem cells have two very special properties: First, unlike other typical mammalian cell types, they can be propagated in culture almost indefinitely while maintaining their pluripotency, providing a virtually unlimited supply. Second, they can be used to generate a variety of tissue types of interest as a source of replacement cells and tissues for use in tissue therapy, or for use in the screening of pharmaceutical agents. Consequently, stem cells are seen as possible sources of chondrocytes.
Kramer et al. (Mech. Dev. 92:193, 2000) reported that mouse embryonic stem cells can be modulated with bone morphogenic proteins (BMP-2 and BMP-4) to produce cells that stained with Alcian blue, a feature of chondrocytes, and expressing the transcription factor scleraxis. However, the mouse model of embryonic stem cell development does not necessarily yield strategies for differentiation that are applicable to other species (see, e.g. Ginis et al. (2004) Dev. Biol 269:360).
Thomson et al. (U.S. Pat. No. 5,843,780; Proc. Natl. Acad. Sci. USA 92:7844, 1995) were the first to successfully isolate and propagate pluripotent stem cells from primates. They subsequently established human embryonic stem (hES) cell lines from human blastocysts (Science 282:114, 1998). Gearhart and co-workers established human embryonic germ (hEG) cell lines from fetal gonadal tissue (Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998; and U.S. Pat. No. 6,090,622). Both hES and hEG cells have the long-sought characteristics of pluripotent stem cells: they can be cultured extensively without differentiating, they have a normal karyotype, and they are capable of producing a number of important cell types including cell types from all three primary germ layers.
Mesenchymal progenitors can be generated from hES cells according to the method described in WO 03/004605. The hES-derived mesenchymal cells can then be further differentiated into osteoblast lineage cells in a medium containing an osteogenic factor, such as bone morphogenic protein (particularly BMP-4), a ligand for a human TGF-β receptor, or a ligand for a human vitamin D receptor (WO 03/004605; Sotile et al., Cloning Stem Cells 2003; 5(2):149-55). Chondrocytes or their progenitors can be generated by culturing hES cells in microaggregates with effective combinations of differentiation factors listed in WO 03/050250.
Hegert et al. (J. Cell Sci. 115:4617, 2002) reported the differentiation plasticity of chondrocytes derived from embryoid bodies composed of mouse embryonic stem cells. Toh et al. describes differentiation and enrichment of expandable chondrogenic cells from human embryonic stem cells in vitro (J. Cell. Mol. Med., 2009—early online publication citation reference 10.1111/j.1582-4934.2009.00762.x).
There remains a need for efficient, scaleable methods capable of producing sufficient quantities of chondrocyte lineage cells, including both chondrocyte progenitors as well as mature chondrocytes for therapeutic and research applications. It would also be useful if a stable chondrocyte precursor could be isolated that could be maintained in culture under scaleable conditions and which could readily be differentiated into mature chondrocytes or cells expressing proteins, such as collagen II, aggrecan and glycosaminoglycans.
However, even with the preparation of chondrocytes derived from undifferentiated progenitor cell populations, such as hES cells, there remains the need to use immunosuppressive drugs in transplantation therapies using chondrocyte cell populations which is unfavourable for the long term success of transplants in patients.
Accordingly, it is necessary to develop a new approach to differentiate primate pluripotent cells into fully functional chondrocytes which avoid the need to use immunosuppressant drugs in transplantation as well as the chondrocyte precursor cells types which could readily give rise to such chondrocytes. It is also necessary to develop a new approach to therapeutically using chondrocytes, such as chondrocytes differentiated in vitro from primate pluripotent stem cells, without the use of immunosuppressive and/or anti-inflammatory agents. It is further necessary to develop a new approach to treating subjects with chondrocytes, such as chondrocytes differentiated in vitro from primate pluripotent stem cells, without the use of immunosuppressive and/or anti-inflammatory agents.